Interkingdom interactions shape the fungal microbiome of mosquitoes

Background The mosquito microbiome is an important modulator of vector competence and vectoral capacity. Unlike the extensively studied bacterial microbiome, fungal communities in the mosquito microbiome (the mycobiome) remain largely unexplored. To work towards getting an improved understanding of the fungi associated with mosquitoes, we sequenced the mycobiome of three field-collected and laboratory-reared mosquito species (Aedes albopictus, Aedes aegypti, and Culex quinquefasciatus). Results Our analysis showed both environment and host species were contributing to the diversity of the fungal microbiome of mosquitoes. When comparing species, Ae. albopictus possessed a higher number of diverse fungal taxa than Cx. quinquefasciatus, while strikingly less than 1% of reads from Ae. aegypti samples were fungal. Fungal reads from Ae. aegypti were < 1% even after inhibiting host amplification using a PNA blocker, indicating that this species lacked a significant fungal microbiome that was amplified using this sequencing approach. Using a mono-association mosquito infection model, we confirmed that mosquito-derived fungal isolates colonize Aedes mosquitoes and support growth and development at comparable rates to their bacterial counterparts. Strikingly, native bacterial taxa isolated from mosquitoes impeded the colonization of symbiotic fungi in Ae. aegypti suggesting interkingdom interactions shape fungal microbiome communities. Conclusion Collectively, this study adds to our understanding of the fungal microbiome of different mosquito species, that these fungal microbes support growth and development, and highlights that microbial interactions underpin fungal colonization of these medically relevent species. Supplementary Information The online version contains supplementary material available at 10.1186/s42523-024-00298-4.


Abstract. 27
The mosquito microbiome is an important modulator of vector competence and vectoral 28 capacity. Unlike the extensively studied bacterial microbiome, fungal communities in the 29 mosquito microbiome (mycobiome) remain largely unexplored. To work towards getting 30 an improved understanding of the fungi associated with mosquitoes, we sequenced the 31 mycobiome of three field-collected and laboratory-reared mosquito species (Aedes 32 albopictus, Aedes aegypti, and Culex quinquefasciatus). Our analysis showed both 33 environment and host species were contributing to the diversity of the fungal microbiome 34 of mosquitoes. When comparing species, Ae. albopictus possessed a higher number of 35 diverse fungal taxa than Cx. quinquefasciatus, while strikingly less than 1% of reads 36 from Ae. aegypti samples were fungal. Fungal reads from Ae. aegypti were <1% even 37 after inhibiting host amplification using a PNA blocker, indicating that this species lacked 38 a significant fungal microbiome that was amplified using this sequencing approach. 39 Using a mono-association mosquito infection model, we confirmed that mosquito-40 derived fungal isolates colonize and for Aedes mosquitoes, support growth and 41 development at comparable rates to their bacterial counterparts. Strikingly, native 42 bacterial taxa isolated from mosquitoes impeded the colonization of symbiotic fungi in 43 Ae. aegypti suggesting interkingdom interactions shape fungal microbiome communities. 44 Collectively, this study adds to our understanding of the fungal microbiome of different 45 mosquito species, that these fungal microbes support growth and development, and 46 highlights that microbial interactions underpin fungal colonization of these medically 47 relevent species. 48 49 Introduction. 50 The microbiome profoundly influences many phenotypes in a host. In mosquitoes, much 51 of the focus in this area has centered on how bacterial microbiota play an important role 52 in mosquito biology, particular in relation to vector competence or how bacteria can be 53 exploited for vector control [1][2][3][4]. Many of these studies have examined how the bacterial 54 microbiome influences mosquito traits important for vectorial capacity, including growth, 55 reproduction, and blood meal digestion [5][6][7][8][9]. While these studies provide convincing 56 evidence that microbes can influence traits important for vectorial capacity of mosquitoes 57 [9,10], the role of the fungi on mosquito biology is understudied and less well 58 understood. 59 60 Several studies have characterized the fungal microbiome in different mosquito species 61 using culture-dependant and -independent methods [11][12][13][14][15][16][17][18][19][20][21][22][23][24][25][26][27]. In general, these studies 62 indicate the majority of fungal taxa that colonize mosquitoes are within the Ascomycota 63 and Basidiomycota phyla [16,19,22,[28][29][30][31]. Shotgun metagenomic sequencing of Cx. 64 pipiens, Culisetra incidens, and Olchelerotatus sierrensis uncovered a diverse array of 65 fungal taxa in mosquitoes, but only two fungal genera, Cladosporium and 66 Chromocliesta, were present in multiple mosquitoes [13]. Amplicon sequencing of 67 bacterial and fungal microbiomes of Ae. aegypti found fewer eukaryotic taxa compared 68 to bacterial, although the majority of eukaryotic reads in mosquitoes were designated to 69 gregarine parasites, rather than fungal species [18]. While our appreciation of the fungal 70 community is expanding, we have a poor understanding of its functional relavance or 71 interactions with other members of the microbiome. 72 Fungal community composition and abundance appear to be influenced by several 73 factors, similar to their bacterial counterparts [28]. Aspects that appear to affect fungal 74 microbiota include habitat, host species, diet, and pathogen infection [16,22,23,30,31]. 75 For instance, in the tree hole mosquitoes Ae. triseriatus and Ae. japonicus, both blood 76 feeding and La Cross virus infection were shown to reduce fungal richness [17]. Like the 77 bacterial microbiome, mycobiome community structure varies between mosquito species 78 and habitats [16][17][18][19]27] and fungal diversity is seen between mosquito tissues [19, 22, 79 30]. While it is evident that mosquitoes possess diverse fungal taxa, sequence based 80 assessment of the fungal microbome can be challenging due to inadvertent amplification 81 of the host. To overcome these challenges, methods to selectively amplify the fungal 82 sequences at the expense of host sequence have been accomplished [11]. Bray-Curtis dissimilarity, P > 0.05). As such, we combined these mosquitoes for further 283 analyses and considered them "field-collected". When comparing between mosquito 284 species, we found that the field-collected Ae. albopictus had significantly elevated 285 Shannon diversity compared to Cx. quinquefasciatus ( Fig. 2A; Tukey's multiple 286 comparison test, P < 0.05), but no difference was seen between species in lab-reared 287 mosquitoes. Similarly, there was no significant difference in Shannon diversity when 288 comparing within a species between environments (i.e. field vs lab; Fig 2A). This was 289 also true for the number of OTUs with no difference within a species but Ae. albopictus 290 had significantly more OTUs compared to Cx. quinquefasciatus regardless of 291 environment ( Fig 2B; Tukey's multiple comparison test, P < 0.05). We then examined 292 the community structure of the fungal microbiome using Bray-Curtis NMDS analysis. 293 Overall, the fungal microbiome clustered distinctly with both species and environment 294 were identified as significant factors ( Fig. 2C; Bray-Curtis dissimilarity, P = 0.0009). This 295 was predominantly driven by the field samples which, when analyzed separately, were 296 significantly different between each species ( fungal OTUs. The fungal diversity was compared between laboratory and field samples 310 between Ae. albopictus and Cx. quinquefasciatus (C). The fungal microbiome in the field 311 collected samples (D) and laboratory reared mosquitoes (E) were compared between 312 the two species. The fungal diversity between different environments (lab v field) was 313 compared within Cx. quinquefasciatus (F) and Ae. albopictus (G). Numbers inside the 314 graph indicates the p-value between groups. The field samples includes mosquitoes 315 were collected in G and BG traps. Key for coloured squares and cicrles is within each 316 panel. The data were analyzed by one-way ANOVA with Tukey's multiple comparison 317 test where P<0.05 considered significant. 318 319 Next, we examined the taxa present in each mosquito species. There were 244 fungal 320 OTUs in mosquitoes, of which 76 and 97 were present above a 0.1% threshold in Cx. 321 quinquefasciatus and Ae. albopictus, respectively (Table S3). While the majority of taxa 322 were unidentified ( Fig. 3 and S6), of the known OTUs, most were classified within the 323 Ascomycota and Basidiomycota phyla, (Fig S6), which was similar to other studies [16, 324 18, 45]. Saccharomycetaceae were the most abundant in Ae. albopictus while the 325 Malasseziaceae where dominant in Cx. quinquefasciatus (Fig 3A and S6). 326 Unsurprisingly, considering the beta diversity analysis, the microbiomes of the lab-reared 327 mosquitoes were comparable, however when examining the diversity between 328 individuals, there was variation (Fig S6), which is also a feature of the bacterial 329 microbiome [35]. In many cases, OTUs that were dominant in one individual were absent 330 or at low abundances from others ( Fig S5). Dunn's multiple comparision test. Growth was determined by percentage of L1 larvae to 390 reach adulthood (D-E). Data were analysed by one-way ANOVA with Tukey's multiple 391 comparision test. None of the axenic larvae pupated and hence, the percentage to 392 adulthood are zero for all axenic controls. 393 394

Fungal infection in presence and absence of native bacterial microbiome. 395
We have previously shown that colonization of symbiotic bacteria is influenced by 396 members of the native bacterial microbiome [35,46]. Given the ability of fungi to infected 397 Ae. aegypti in a mono-association but the lack of fungal reads in field-collected 398 mosquitoes, we speculated that bacteria may inhibit fungal infection. To determine if 399 cross kingdom interactions influenced fungal colonization, we infected fungi into 400 conventionally reared or axenic Ae. aegypti, which either possessed or lacked their 401 native bacterial microbiome, respectively. Strikingly, we did not recover any fungal CFUs 402 in either larvae or adults when the mosquitoes were grown conventionally in the 403 presence of a native microbiome, however in stark comparison, fungal isolates were 404 able to effectively colonize germ-free mosquitoes (Fig. 6, Mann Whitney Test, P<0.05). 405 Intringuingly, the reduced colonization capacity of fungi of conventionally reared 406 mosquitoes was seen in both larvae (Fig. 6A, Mann Whitney Test, P<0.05) and adults 407 ( Fig. 6B, Mann Whitney Test, P<0.05). In agreement with our previous study [38], the 408 positive control, C. neteri also was more effective at colonizing germ-free mosquitoes 409 compared to their conspecfic's that possessed a conventional microbiome, however this 410 effect here was more subtle compared to the almost complete blockage of fungi seen 411 when mosquitoes had bacterial microbiota. Ae. aegypti mosquitoes that possessed their native microbiota or axenic germ-free 416 mosquitoes to create a mono-association (MA). CFUs were quantified in A) L2-L3 larvae 417 and B) three to four day old adults. The bacterium C. neteri was used as a positive control.

418
A contamination control was undertaken by rearing axenic larvae without infection. These 419 mosquitoes did not develop confirming sterility. The CFU/mosquito data were analysed by 420 unpaired t test and prevalence data by a Fishers exact test. Asterisks (*) indicates 421 significance, while ns denotes non-significant. 422 423

Discussion. 424
We characterised the fungal microbiome of Ae. aegypti, Ae. albopictus and Cx. 425 quinquesfaciatus collected from different environments. Sufficient fungal reads were 426 obtained from Cx. quinquesfaciatus and Ae. albopictus to evaluate their fungal 427 microbiomes. In these species, we found the fungal composition varied substantially 428 between species and environments. These findings were similar to other reports 429 whereby environment has been shown to be a major determinant of fungal microbiome 430 composition [16,18,19]. At the individual level, there was variability in the composition 431 of fungal taxa within mosquitoes. Of the known taxa, Malassezia, Saccharomcetales, 432 and to a lesser extent, Candida were fungi that were frequently seen in either species 433 and other studies have identified these genera in mosquitoes suggesting they may 434 commonly infect these vectors [13,16,29,47,48]. 435 436 Strikingly, our sequencing data suggest that the fungal microbiome of Ae. aegpyti is 437 dramatically reduced as we only observed a small fraction of fungal reads in these 438 mosquitoes. Initially we speculated that the low number of fungal reads was due to 439 preferential amplification of the host, and as such we used blocking PNA 440 oligonucleotides to suppress host reads, in a similar fashion to other studies [11,43,44]. 441 Despite our blocking primer reducing host ITS reads, there was no significant increase in 442 the number of fungal reads, but rather an increase in off target host reads, indicating that 443 these field caught mosquitoes lacked fungi at an amplifiable level. Supporting this 444 finding, qPCR analysis of lab-reared Ae. aegypti found significantly reduced fungal 445 densities compared to Ae. albopictus and Cx. quinquesfaciatus. Together these data 446 indicate that these Ae. aegypti mosquitoes have a reduced fungal microbiome. Further 447 studies are required to determine if this is consistent across other lab-reared or field 448 collected Ae. aegypti mosquitoes. 449

450
Little is known about the capacity of members of the fungal microbiome to colonize their 451 mosquito host. Although our sequencing data indicate Ae. aegypti lacked a robust fungal 452 microbiome, specific taxa were able to colonize when infected into germ-free 453 mosquitoes. The ability of germ-free mosquitoes to harbour fungi suggests that the 454 reduced fungal load that we saw in Ae. aegypti by sequencing or qPCR was not due to 455 an incompatibility between the fungal species and the mosquito, but rather due to 456 microbial incompatibility. To empirically test this, we compared infection of fungal taxa in 457 germ-free compared to conventially reared mosquitoes and found fungi infected the 458 mosquitoes in absence of native microbiome. While the microbiome can be composed of 459 a variety of microbes, we speculated that bacterial microbiota were interfering with 460 fungal infections. We have previously identified several bacterial co-occurrence 461 interactions in these mosquitoes and experimentally validated inter-bacterial interactions 462 in co-infection studies [35,46,49]. However, fungal-bacterial co-occurance has not been 463 exclusively investigated. Several other studies identified fungal and bacterial 464 communities co-existing from individual mosquitoes, but these were not in Ae. aegypti 465 show that native fungal species that associate with mosquitoes also have the ability to 474 support mosquito growth and development. We did observed developmental variation 475 between fungal microbes and between mosquito species, however, S. cerevisiae had 476 similar developmental rates compared to previous studies [23,55]. Interestingly, we saw 477 variability between replicates in terms of S. cerevisiae infections. These replicate tempting to speculate that differences in the native microbiota were responsible for the 484 variation in S. cerevisiae colonization. These findings will be important to confirm given 485 that S. cerevisiae is being investigated for novel vector control strategies [57]. 486

487
In summary, here we showed that Ae. albopictus and Cx. quinquefasciatus harbor 488 fungal taxa as part of their microbiome, but, Ae. aegypti appear to lack mycobiome. The 489 lack of fungal taxa in Ae. aegypti appears to be due to cross kingdom microbial 490 interactions. Despite this, when the bacterial microbiome is removed, fungi can infected 491 these mosquitoes and support their growth. Together, our findings have shed a light on 492 an understudied aspect of the mosquito microbiome and shown that native fungal 493 symbionts influence mosquito biology.  Table S1. Sequences of PCR primers used in the study 497

Acknowledgements. 530
We would like to thank the UTMB insectary core for providing the lab mosquitoes. GLH 531 was supported by the BBSRC (BB/T001240/1, BB/V011278/1, BB/X018024/1, and 532 BB/W018446/1), the UKRI (20197 and o  u  a  c  h  e  K  ,  M  a  r  t  i  n  E  ,  R  a  h  o  l  a  N  ,  G  a  n  g  u  e  M  F  ,  M  i  n  a  r  d  G  ,  D  u  b  o  s  t  A  ,  e  t  a  l  .  L  a  r  v  a  l  h  a  b  i  t  a  t   633   d  e  t  e  r  m  i  n  e  s  t  h  e  b  a  c  t  e  r  i  a  l  a  n  d  f  u  n  g  a  l  m  i  c  r  o  b  i  o  t  a  o  f  t  h  e  m  o  s  q  u  i  t  o  v  e  c  t  o  r  A  e  d  e  s  a  e  g  y  p  t